Aquatic birnavirus-induced ER stress-mediated death signaling contribute to downregulation of Bcl-2 family proteins in salmon embryo cells

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.     

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.Subject terms: Cell biology, Cancer     

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a γ-secretase inhibitor, N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes α- and γ-secretase cleavage and subsequently induces CHOP-mediated cell death.     

Misfolded proteins inhibit proliferation and promote stress-induced death in SV40-transformed mammalian cells

Protein misfolding is implicated in neurodegenerative diseases and occurs in aging. However, the contribution of the misfolded ensembles to toxicity remains largely unknown. Here we introduce 2 primate cell models of destabilized proteins devoid of specific cellular functions and interactors, as bona fide misfolded proteins, allowing us to isolate the gain-of-function of non-native structures. Both GFP-degron and a mutant chloramphenicol-acetyltransferase fused to GFP (GFP-Δ9CAT) form perinuclear aggregates, are degraded by the proteasome, and colocalize with and induce the chaperone Hsp70 (HSPA1A/B) in COS-7 cells. We find that misfolded proteins neither significantly compromise chaperone-mediated folding capacity nor induce cell death. However, they do induce growth arrest in cells that are unable to degrade them and promote stress-induced death upon proteasome inhibition by MG-132 and heat shock. Finally, we show that overexpression of all heat-shock factor-1 (HSF1) and Hsp70 proteins, as well as wild-type and deacetylase-deficient (H363Y) SIRT1, rescue survival upon stress, implying a noncatalytic action of SIRT1 in response to protein misfolding. Our study establishes a novel model and extends our knowledge on the mechanism of the function-independent proteotoxicity of misfolded proteins in dividing cells.     

Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression

Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells.     

A Highlights from MBoC Selection: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop^−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK''s down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.     

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.     

TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death

C/EBP homologous protein (CHOP) is a stress-inducible nuclear protein that is crucial for the development of programmed cell death and regeneration; however, the regulation of its function has not been well characterized. Slbo, a Drosophila homolog of C/EBP (CCAAT/enhancer binding protein), was shown to be unstabilized by tribbles. Here, we identified TRB3 as a tribbles ortholog in humans, which associated with CHOP to suppress the CHOP-dependent transactivation. TRB3 is induced by various forms endoplasmic reticulum (ER) stress later than CHOP. Tunicamycin treatment enhanced the TRB3 promoter activity, while dominant-negative forms of CHOP suppressed the tunicamycin-induced activation. In addition, the tunicamycin response region in the TRB3 promoter contains amino-acid response elements overlapping the CHOP-binding site, and CHOP and ATF4 cooperated to activate this promoter activity. Knockdown of endogenous ATF4 or CHOP expression dramatically repressed tunicamycin-induced TRB3 induction. Furthermore, knockdown of TRB3 expression decreased ER stress-dependent cell death. These results indicate that TRB3 is a novel target of CHOP/ATF4 and downregulates its own induction by repression of CHOP/ATF4 functions, and that it is involved in CHOP-dependent cell death during ER stress.     

Cytotoxic mechanisms of panduratin A on A375 melanoma cells: A quantitative and temporal proteomics analysis

Melanoma is a lethal form of skin cancer with rising global incidence. However, limited treatment options are available for advanced melanoma and this is further compounded by the development of resistance toward existing drugs. Panduratin A (PA), a cyclohexanyl chalcone found in Boesenbergia rotunda, was investigated for its cytotoxic potentials against human malignant melanoma A375 cells. Our initial findings revealed that mitochondrion is the primary acting site of PA on A375 cancer cells and the cytotoxic mechanisms of PA were further investigated using a temporal quantitative proteomics approach by iTRAQ 2D‐LC‐MS/MS. Comprehensive proteomics analysis identified 296 proteins that were significantly deregulated in PA‐treated A375 cells and revealed the involvement of mitochondrial oxidative phosphorylation, secretory and ER stress pathway, and apoptosis. We further confirmed that the PA‐induced apoptosis was mediated by prolonged ER stress at least in part via the PERK/eIF2α/ATF4/CHOP pathway. Pretreatment with cycloheximide, an ER stress inhibitor rescued PA‐induced cell death, which was accompanied by the suppression of ER‐stress‐related HSPA5 and CHOP proteins. The present study provides comprehensive mechanistic insights into the cytotoxic mechanisms of PA.     

The Unfolded Protein Response Transducer ATF6 Represents a Novel Transmembrane-type Endoplasmic Reticulum-associated Degradation Substrate Requiring Both Mannose Trimming and SEL1L Protein

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.